Molecular Structure of SPB Core Proteins

SPB核心蛋白的分子结构

基本信息

批准号：
8668222
负责人：
IVAN RAYMENT
金额：
$ 31.74万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8668222
关键词：
Address Air Biochemical Genetics Biological C-terminal Calmodulin Cells Cellular biology Centrosome Complement Complex Core Protein Cryoelectron Microscopy Data Set Docking Exhibits Fluorescence Resonance Energy Transfer Goals In Situ Individual Investigation Knowledge Location Maps Measurement Measures Mechanics Methodology Microtubule-Organizing Center Microtubules Modeling Molecular Molecular Models Molecular Structure Mutation Nature Nuclear Envelope Phosphorylation Positioning Attribute Property Proteins Regulation Research Personnel Resolution Roentgen Rays Structural Models Structure Tertiary Protein Structure Testing Time Tomogram Tubulin X-Ray Crystallography Yeast Model System Yeasts base crosslink detector electron tomography in vivo insight molecular modeling novel reconstruction restraint self assembly skills success three-dimensional modeling

Address Air Biochemical Genetics Biological C-terminal Calmodulin Cells Cellular biology Centrosome Complement Complex Core Protein Cryoelectron Microscopy Data Set Docking Exhibits Fluorescence Resonance Energy Transfer Goals In Situ Individual Investigation Knowledge Location Maps Measurement Measures Mechanics Methodology Microtubule-Organizing Center Microtubules Modeling Molecular Molecular Models Molecular Structure Mutation Nature Nuclear Envelope Phosphorylation Positioning Attribute Property Proteins Regulation Research Personnel Resolution Roentgen Rays Structural Models Structure Tertiary Protein Structure Testing Time Tomogram Tubulin X-Ray Crystallography Yeast Model System Yeasts base crosslink detector electron tomography in vivo insight molecular modeling novel reconstruction restraint self assembly skills success three-dimensional modeling

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项目摘要

The overall goal of this project is to establish the experimental restraints necessary to construct a molecular model for SPB core that extends from atomic structures of the individual components to their interactions with each other and their overall position in the MTOC where the information will be assembled into a cohesive structure by the computational core. This structural study complements the investigation of the structure of the y-tubulin complexes that constitute the inner and outer plaques that will be performed by Project 2 (Agard). The experimental plan includes three complementary approaches. The first aim (Rayment) begins with high resolution X-ray structural determinations of the individual components of the SPB and proceeds in a logical progression from the structures of the proteins and domains that constitute the central plaque which is embedded in the nuclear envelope (Spc110-C, calmodulin, Spc29, and Spc42-N) through a structural determination of the Intermediate layers 2 and 1 (Spc42-C, Cnm67, Nud1, and Spc72-C). Intermediate Layer 1 forms the bridge to the y-tubulin (Tub4) complex in the outer plaque so that this project interfaces and complements the study of the y-tubulin described in Project 2. At this time all of the proteins in the SPB core have been expressed in a soluble form suitable for structural or biophysical study and more than half of them have been crystallized. The second aim is directed towards establishing a molecular envelope for the native SPB into which the high-resolution structures can be docked or modeled (Agard). This will be established through cryo electron tomography (cryoET) and subvolume averaging of entire isolated SPB. This will provide an unbiased 3D framework of the entire SPB at an intermediate resolution of about 20 Å. It will reveal the domain organization within each layer and the interaction of major SPB components between layers. Protein-tagging will be used to locate and orient individual proteins within the maps. The third aim (Davis) is directed towards generating a new set of distance restraints between individual components that are needed to combine the information from the previous specific aims. Two approaches will be used. First, the prior FRET analysis that established the current arrangement of SPB components in the core will be extended for proteins in the central plaque. Second, a new set of high-resolution distances will be established through crosslinking analysis of native SPB assemblies. These structural investigations will provide detailed structures of the individual components and the manner in which they are arranged in the SPB. Thus, it will establish air of the information necessary for the computational core to generate a pseudo atomic model for the SPB. In turn, hypotheses that arise from this three-dimensional model will be tested through biochemical, genetic and cell biological studies in Projects 3 and 4 (Davis, Winey and Rayment). It will also allow the mechanical strengths of the microtubule-SPB attachments measured in Project 5 (Asbury) to be interpreted in molecular terms.

该项目的总体目标是建立为SPB核心构建分子模型所需的实验限制，该模型从单个组件的原子结构扩展到彼此之间的相互作用以及它们在MTOC中的总体位置，在该MTOC中，信息将通过计算核心将信息组装到凝聚结构中。这项结构性研究符合Y构成项目2（AGARD）将执行的内部和外部斑块的Y微管蛋白复合物结构的投资。实验计划包括三种完整的方法。第一个目的（射线）首先是对SPB单个组件的高分辨率X射线结构确定，并从构成构成中心斑块的蛋白质和域的结构中进行逻辑进展，该蛋白质和域的结构嵌入了核包膜中（SPC110-C，Calsodulin，spc29，SPC29，以及callodins spc29，spc42-n）的层次，并进行。（SPC42-C，CNM67，NUD1和SPC72-C）。 1层中间层形成了外部斑块中Y-微管蛋白（TUB4）复合物的桥梁，因此该项目接口和综合项目2中描述的Y-微管蛋白的研究。此时，SPB核心中的所有蛋白质都以固体形式表达，适用于结构或生物物理学研究，而其中一半的蛋白质以及其中的一半是结晶的。第二个目的是为了建立一个可以对接或建模高分辨率结构的天然SPB的分子包膜（AGARD）。这将通过冷冻电子断层扫描（冷冻）和整个孤立的SPB的平均。这将提供整个SPB的无偏3D框架，中间分辨率约为20Å。它将揭示每一层中的域组织以及层之间主要的SPB组件的相互作用。蛋白质标记将用于在地图中定位和定向单个蛋白质。第三个目标（戴维斯）是针对在需要将信息结合到以前的特定目标中所需的各个组件之间产生一组新的距离限制。将使用两种方法。首先，以前的FRET分析确定了核心中SPB组件的当前布置，将扩展到中央斑块中的蛋白质。其次，将通过对天然SPB组件的交联分析来建立一组新的高分辨率距离。这些结构性投资将提供各个组件及其在SPB中排列的方式的详细结构。这将建立计算核心为SPB生成伪原子模型所必需的信息的空气。反过来，这三维模型将通过项目3和4（戴维斯，葡萄酒和雷神）中的生化，遗传和细胞生物学研究来检验。它还将允许在项目5（Asbury）中测得的微管SPB附着的机械强度以分子术语来解释。